Autoimmunity is a prevalent problem in the United States, affecting more than 8 million Americans. Understanding how changes in the immune system can result in autoimmunity is an important step in finding better ways to diagnose and treat patients with autoimmune disease. The long-term objective of this project is to understand the pathophysiology of autoimmune diseases by studying a novel mouse model of spontaneous autoimmunity, the G228W mouse, which has a point mutation in Autoimmune regulator (Aire). Aire is important in preventing autoimmune disease. Humans and mice deficient in Aire develop organ- specific autoimmune disease in an autosomal recessive manner. Mice deficient in Aire have decreased ectopic expression of certain thymic self-antigens (for example, insulin), and a concomitant impairment of T cell negative selection in the thymus. These results are consistent with a model in which Aire increases expression of certain organ-specific self-antigens in the thymus, and recognition of these self-antigens by developing T cells results in deletion of these self-reactive cells. The exact mechanism by which Aire acts to drive thymic expression of self-antigens is not well understood. To address this question, I have developed the G228W mouse model which harbors a knockin point mutation in a predicted DNA binding domain of Aire. Like people with this mutation, this mouse model develops autosomal dominant autoimmunity in a distinct set of organs. This novel mouse model will be a powerful tool to study the structure-function relationship in Aire. The hypothesis for this proposal is that this single amino acid change in Aire interferes with binding to the promoter regions of thymic self-antigens that are important in the pathogenesis of autoimmune disease. The three goals for this proposal are to: 1) identify thymic self-antigens that are differentially regulated in the G228W mouse, 2) identify thymic self-antigens important in causing disease in the G228W mouse, and 3) determine the effect of the G228W mutation on DNA binding to the promoter regions of antigens important in causing disease. Understanding the mechanism by which Aire functions has important implications for how autoimmune diseases are diagnosed and treated.